Cancer is one of the leading causes of death in industrialized countries. In the United States, cancer is the second leading cause of all deaths and accounts for hundreds of thousands of deaths each year. Cancer is a devastating disease on many levels. For example, gliomas are the main cause of death of patients with brain tumors. Patients with glioblastoma have a mean survival time of less than 12 months, and this prognosis has not changed much since 1959 (ten months) and 1932 (six to nine months) despite impressive developments in methods for treating cancer.
Cancer may be treated by a variety of methods including surgery, radiotherapy, chemotherapy, and immunotherapy. Although these methods of treatment have in general improved the survival rates of cancer victims, the fact remains that there is a clear need for improved therapeutic techniques for combating cancer.
A resurgent approach to treating cancer is referred to as virotherapy. Virotherapy first garnered the interest of scientists when it was discovered that tumors of some cancer patients regressed after they experienced viral infection or vaccination (see the reviews of Sinkovics, J., & Horvarth, J. [1993] Intervirology 36:193–214 and Alemany et al. [2000] Nat. Biotech. 18:723–727). Unfortunately, the initial promise of this approach was diminished when researchers discovered toxicity problems associated with virotherapy and furthermore the treatments had limited efficacy.
The advent of modern molecular biology has prompted scientists to reassess the feasibility of virotherapy. In particular, virus mediated gene therapy is now the central focus in the renewed interest in virotherapy. The molecular strategy underlying the design of virus mediated gene therapy systems is to deliver a gene which will inhibit tumor cell growth (e.g., controlling cell cycle or apoptosis), kill the cell (suicide gene), or induce an immune response (immunotherapy).
Two general approaches are available. One approach has centered on the use of replication-deficient viral vectors. The use of replication deficient vectors as virotherapeutic agents has encountered two major problems: (1) low in vivo transduction efficiency, resulting in poor gene delivery and (2) inability to specifically target tumor versus normal tissue.
Another approach to virotherapy involves the use of replication-competent viruses. The use of replication-competent viruses with a cytolytic cycle, has emerged as a viable strategy for directly killing tumor cells (oncolysis) as well as enhancing gene transfer and specifically targeting tumor cells. A variety of modified neuroattenuated herpes simplex viruses (HSVs) with deletions in the genes for neurovirulence (y134.5) and ribonucleotide reductase (UL39), function as oncolytic agents for human tumor cells in vitro and in mouse models of human brain tumors in vivo. See, e.g., Parker et al. (2000) PNAS 97:2208–2213 and U.S. Pat. No. 5,728,379. In phase 1 clinical trials, twenty-one patients with malignant glioma have received intracranial injections of HSV G207 without any signs of encephalitis or CNS changes (Markert et al. [2000] Gene Ther. 10:867–874).
A different strategy makes use of an oncolytic, replication competent adenovirus (dl1520/ONYX-015) which has a deletion that leads to abrogated production of the 55 kDa E1B protein (Bischoff et al. [1996] Science 274:373–376; U.S. Pat. Nos. 6,080,578 and 5,677,178). Preliminary data suggested that the replication of this virus was restricted to tumor cells with a deficient p53 tumor suppressor gene. However, more recent findings have established that cells which are wild type for p53 gene status can also support replication of this virus (Rothmann et al. [1998] J. Virol. 72:9470–9478; Goodrun & Ornelles [1998] J. Virol. 72:9479–9490). This virus is currently being used in phase 1 clinical trials for ovarian cancer and gastrointestinal cancers that have metastasized to the liver, as well as in phase 2 and 3 clinical trials for recurrent and refractory head & neck cancer. Results so far have shown that injection of replication-competent adenovirus dl1520 is safe and well-tolerated by patients, whose complaints are mainly minor grade 1–2 flu-like symptoms (Kim [2000] Oncogene 19:6660–6669). The low toxicity of these two viral systems in humans suggests that replication-competent viruses are promising approaches for treating patients with tumors. More recently, the design of oncolytic viruses whose replication is restricted to a specific tumor type has been realized. An adenovirus (CN706) was created which showed a selective cytotoxicity for prostate-specific antigen (PSA) positive cancer cells in vitro and in murine prostate cancer models in vivo (Rodriguez et al. [1997] Cancer Res. 57:2559–2563; U.S. Pat. Nos. 5,871,726 and 6,197,293).
While such approaches utilizing replication-competent viruses are promising, they are limited in that: (1) multiple viruses would have to be created for different tumor types and possibly individual tumors due to the genetic heterogeneity of tumors, (2) they do not provide for the selective targeting of tumors derived from a broad range of tissues, and (3) they potentially require rigorous anti-viral treatments to eliminate virus after completion of therapy.
A multitude of U.S. patents have issued regarding hypoxia-inducible factor-1, virus mediated gene delivery, tissue specific constructs, and related topics.
Several patents to Semenza and Semenza et al. relate to hypoxia-inducible factor-1. U.S. Pat. No. 6,222,018 to Semenza discloses a substantially purified hypoxia-inducible factor (HIF-1) characterized as being capable of activating gene expression in genes that contain a HIF-1 binding site. U.S. Pat. No. 6,124,131 to Semenza discloses a substantially purified stable human hypoxia-inducible factor-1α as well as nucleotides encoding the same. U.S. Pat. No. 5,882,914 to Semenza discloses nucleic acids encoding hypoxia inducible factor-1 as well as purification and characterization of the expressed proteins.
The patents to Semenza and Semenza et al. refer to purified HIF-1, nucleic acids encoding HIF-1, antibodies that bind HIF-1, mutants of HIF-1, and method of using all of these biological molecules. The patents do not describe recombinant viruses that selectively replicate in and cytolyse hypoxic/HIF-active tissue and have the capability of delivering an anti-angiogenic factor or other proteins with anti-tumor activity. U.S. Pat. No. 6,218,179 to Webster et al. discloses tissue-specific hypoxia regulated constructs. Webster et al. describes a method for reducing ischemic injury to a cell exposed to hypoxic conditions. The constructs for reducing ischemic injury described in Webster et a comprise a chimeric gene containing a hypoxia responsive element, a therapeutic gene and a tissue-specific promoter. The therapeutic gene is selected so that its expression is effective in reducing ischemic injury to the cell. Examples of therapeutic genes are those for nitric oxide synthase, Bcl-2, superoxide dismutase and catalase. U.S. Pat. No. 5,834,306 to Webster et al. discloses a method and compositions comprising chimeric genes. The chimeric genes contain a tissue-specific promoter and a hypoxia responsive enhancer element, both of which are operably linked to a selected gene.
Recombinant adeno-associated virions and methods of using them are described in a series of patents to Podsakoff et al. U.S. Pat. No. 6,211,163 to Podsakoff et al. discloses methods for delivering DNA to the bloodstream using recombinant adeno-associated virus vectors. The invention is based on the discovery that recombinant adeno-associated virions are efficiently delivered to various muscle cell types and provide for the sustained production of therapeutic proteins. U.S. Pat. No. 5,858,351 to Podsakoff et al. discloses the use of adeno-associated virus virions for delivering DNA molecules to muscle cells and tissues. U.S. Pat. No. 5,846,528 to Podsakoff et al. discloses recombinant adeno-associated virus virions for delivering DNA molecules to muscle cells and tissues in the treatment of anemia. The Podsakoff et al. patents do not describe recombinant viruses which cytolyse hypoxic/HIF-active tissue and provide an anti-angiogenic factor or other proteins with anti-tumor activity.
U.S. patents related to the hypoxia-inducible factor-1 pathway describe a number of strategies. U.S. Pat. No. 6,184,035 to Csete et al. discloses methods for isolating, activating, and controlling differentiation from skeletal muscle stem or progenitor cells by using hypoxic conditions. The patent to Csete et al. relates to the discovery that adult skeletal muscle fibers cultured under hypoxic conditions give rise to greater numbers of progenitor cells as compared to muscle fibers grown under normal oxygen levels. U.S. Pat. No. 6,130,071 to Alitalo et al. discloses purified and isolated vascular endothelial growth factor-C cysteine deletion variants. U.S. Pat. No. 5,952,226 to Aebischer et al. discloses a device and method for delivery of EPO to a patient using an implanted device that continuously releases EPO. The invention described in the Aebischer et al. patent relates to providing EPO to a subject with cells engineered to express high levels of EPO under hypoxic conditions. U.S. Pat. No. 5,681,706 to Anderson et al. discloses genetic regulatory elements which effect anoxic induction of a DNA molecule in mammalian cells exposed to anoxia. U.S. Pat. No. 5,942,434 to Ratcliffe et al. discloses nucleic acid constructs comprising hypoxia response elements operably linked to a coding sequence such as genes for pro-drug activation systems or cytokines. As seen from these patents, the hypoxia-inducible pathway was harnessed for use in a specific context, recombinant viruses which selectively replicate in and cytolyse hypoxic tissue, or tissues with an activated HIF pathway, and further deliver adjuvant therapy, are not described in the prior art.
The following U.S. patents are expressly incorporated herein by reference to the extent that they are not inconsistent herewith: U.S. Pat. Nos. 6,222,018; 6,218,179; 6,211,163; 6,184,035; 6,130,071; 6,124,131; 5,952,226; 5,942,434; 5,882,914; 5,858,351; 5,846,528; 5,834,306, and 5,681,706.
Thus, there is a need for virotherapeutic systems which combine a therapeutic gene delivery approach and an oncolytic mechanism for the selective targeting of a wide variety of tumors.
A variety of particular problems were encountered during the discovery of this invention. Infection of cells by viruses is a complicated biochemical process. It was first necessary to show that tumor cells could be infected by a recombinant adenovirus under hypoxia. Next, hypoxia-induced expression of constructs in transfected tumor cell lines had to be demonstrated. The HIF-activated expression also had to have an appropriate O2 concentration versus expression level profile. Hypoxia inducible constructs that bi-directionally express gene products had to be designed and tested for embodiments of the invention that involve delivery of adjuvant therapy. After these initial stages of designing and testing, recombinant viruses which contained the constructs were examined in transfected tumor cell lines for expression of E1A and E1B gene products. These expression studies demonstrated that hypoxia-dependent regulation seen in the transient reporter gene assay was maintained in the context of the viral genome. The next step in the invention involved demonstrating that the recombinant virus cytolyzed tumor cells in a hypoxia/HIF-active-dependent manner. Lastly, the inventors showed that the recombinant virus was delivered to brain tumors in a model system. Finally, these studies led to the recombinant virus of the invention.